Measurements of the BKCa channel's high-affinity Ca2+ binding constants: effects of membrane voltage.
It has been established that the large conductance Ca(2+)-activated K(+) channel contains two types of high-affinity Ca(2+) binding sites, termed the Ca(2+) bowl and the RCK1 site. The affinities of these sites, and how they change as the channel opens, is still a subject of some debate. Previous estimates of these affinities have relied on fitting a series of conductance-voltage relations determined over a series of Ca(2+) concentrations with models of channel gating that include both voltage sensing and Ca(2+) binding. This approach requires that some model of voltage sensing be chosen, and differences in the choice of voltage-sensing model may underlie the different estimates that have been produced. Here, to better determine these affinities we have measured Ca(2+) dose-response curves of channel activity at constant voltage for the wild-type mSlo channel (minus its low-affinity Ca(2+) binding site) and for channels that have had one or the other Ca(2+) binding site disabled via mutation. To accurately determine these dose-response curves we have used a series of 22 Ca(2+) concentrations, and we have used unitary current recordings, coupled with changes in channel expression level, to measure open probability over five orders of magnitude. Our results indicate that at -80 mV the Ca(2+) bowl has higher affinity for Ca(2+) than does the RCK1 site in both the opened and closed conformations of the channel, and that the binding of Ca(2+) to the RCK1 site is voltage dependent, whereas at the Ca(2+) bowl it is not.
Sweet
Tara-Beth
TB
Molecular Cardiology Research Institute, Tufts Medical Center, Tufts University School of Medicine, Boston, MA 02111, USA.
Cox
Daniel H
DH
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D020013:Calcium Signaling; D002461:Cell Line, Transformed; D004560:Electricity; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D007700:Kinetics; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure*; D008564:Membrane Potentials / Q000502:physiology*; D051379:Mice; D050505:Mutant Proteins / Q000502:physiology; D018408:Patch-Clamp Techniques; D013329:Structure-Activity Relationship
D051036:Large-Conductance Calcium-Activated Potassium Channels; D050505:Mutant Proteins; D002118:Calcium